To gain more insight into the various functions of EphA4 receptor during the
development of the central nervous system (CNS), we have characterized its cellular and
subcellular localization in the rat hippocampus, first in the adult, and second during the
postnatal development. We have also examined its potential roles in the genesis, migration,
or maturation of the granule cells in the adult hippocampus. For that purpose, we have used
immunocytochemistry in light, electron, and confocal microscopy.
At the light microsocpic level, a strong EphA4 immunoreactivity (peroxidase/DAB)
is observed at postnatal days 1 and 7 (P1 and P7) in the cell body layers, with a labeling
notably associated with the surface of pyramidal and granule cell bodies, as well as in the
neuropil layers of CA3, CA1, and dentate gyrus regions. The intensity of the labeling
diminishes progressively in the cell body layers, between P7 and P14, to become weak at
P21 and in the adult, while it persists in the neuropil layers, except in those receiving inputs
from the entorhinal cortex. At the electron microscopic level, after peroxidase/DAB
labeling, EphA4 covers the entire surface of pyramidal and granule cells, from the cell body
to the distal extremities, between P1 and P14, but becomes restricted to the synaptic
extremities, i.e. the axon terminals and dendritic spines, at P21 and in the adult. At the
plasma membrane of astrocytes, EphA4 is redistributed as in neurons, from the cell body
and proximal to distal processes, at P1 and P7, to the distal perisynaptic processes, at P14
and older ages. In addition, axons in the process of myelination present strong punctiform
immunoreactivity at their plasma membrane, at P14 and P21. Moreover, in neurons and
astrocytes, the endoplamic reticulum, Golgi apparatus, and transport vesicles, organelles
involved in the synthesis, post-translational modifications, and transport of glycosylated
proteins, are also labeled, and also more intensely in younger animals. Lastly, EphA4 is
located in the cell body and dendrites of adult-generated granule cells, at the stage of
maturation where they express doublecortin (DCX). In addition, EphA4 adult knockout
mice display DCX-positive granule cells in an ectopic position, outside of the subgranular
zone, suggesting a role for EphA4 in the regulation of their migration.
This work thus reveals a redistribution of EphA4 in neuronal and glial cells, in the
cellular sites where cellular motility occurs during their maturation: the cell bodies when
they position and organize themselves into layers, the dendritic and axonal processes during
their growth, guidance, and maturation, and the dendritic spines, axon terminals, and distal
astrocytic processes when synapses are formed or strengthened. These locations could thus
reflect different roles for EphA4, similarly associated with the regulation of plasticity in the
CNS, according to the stage of development, the region, the CNS integrity, or the
behavioural experience of an animal.